Cytokines play a central role in regulating neoplastic cell growth/n v/vo. They exert such effects either by a direct induction of growth suppressor genes in the tumor or by mounting an immune response against it. In particular, the interferon family of cytokines regulates several physiologic responses such as antiviral, antitumor, and immune functions. By interacting with other cytokines, IFNs form a large network of intercellular signaling molecules that control neoplastic cell growth and host defenses against pathogens. Among their various actions, the regulation of growth suppression by IFNs is not fully understood. In an earlier study, we demonstrated that the IFN-induced anti-tumor actions, especially apoptosis, could be synergistically augmented upon combination with retinoic acid. This vitamin-A metabolite by itself acts as a growth inhibitor in many experimental models. Some clinical studies also demonstrated the potent tumor inhibitory effects of IFN/RA. In the light of these data, it is important to understand the mechanisms of action of the molecular regulators that cause tumor growth suppression. We hypothesized that IFN/RA employs either novel gene products for inducing cell death. To identify the critical molecules involved in IFN/RA-induced apoptosis, we have taken a genetic approach that identifies genes on the basis of their functions. Here, we have characterized a novel gene product, (gene associated with Retinoid-lnterferon induced Mortality-19 (GRIM-19), which induces apoptosis. The anti-oncogenic effects of GRIM-19 were further highlighted in our recent studies, where its apoptotic functions were suppressed by DNA viral oncoproteins. GRIM-19-expression appears to be inhibited in some human tumors. These characteristics indicate its potent anti-oncogenic characteristics. To gain a better insight into the mechanisms of action, we have used a yeast-2-hybrid screen and identified Signal Transducing Activator of Transcription-3 (STAT3) as a GRIM-19 binding protein. Several recent studies have shown that STAT3, an otherwise dormant signal-regulated transcription factor, is constitutively activated in human tumors. A number of viral and cellular oncoproteins constitutively stimulate its activity to promote cell survival. Based on our preliminary observations, we have hypothesized that GRIM-19 inhibits STAT3-regulated transcription. In this proposal we will investigate the mechanisms of GRIM-19 induced inhibitory effects on tumor cell growth. In particular, we will study the anti-oncogenic effects of GRIM-19 on a constitutively active STAT3 variant, and an oncogenic Src, which uses STAT3 for promoting cell growth. We will also define the critical domains that mediate GRIM-19:STAT3 interactions. Using chromatin immunoprecipitation (CHIP) assays, STAT3-regulated reporter genes and mutant cell lines lacking STAT3, we will investigate the mechanisms of the anti-STAT3 effects of GRIM-19. GRIM-19 may be: 1) a novel tumor suppressor; 2) serve as a marker for the tumor response to therapeutic agents 3) inhibit several other cancer cells by participating in other growth suppressive pathways; 4) be useful in gene therapy once its mode of action has been defined. Thus, an understanding of GRIM-19 action will further help in defining the tumor cell-specific dysfunctions that ablate this new growth regulatory pathway. In summary, the studies proposed in this application will uncover a novel mechanism of cell growth regulation employed by a newly discovered anti-oncogenic protein.